PROJECT SUMMARY Caspases are cysteine proteases that control apoptotic cell death. If caspases are activated, cancer cells die; conversely, inhibiting caspases can prevent cell death in diseases such as heart attack and stroke. Thus, there has been significant interest in caspases as drug targets. This interest heightened further when caspase-6 was discovered to play a central role in neurodegeneration. Unfortunately, to date, no caspase- directed therapies are on the market, primarily because work has focused on the active site, which is the most overlapping and conserved region of the family. It is becoming increasingly clear that each caspase is regulated in a unique and nuanced manner, so the only hope for achieving caspase-specific inhibition is by harnessing their regulation, which is usually mediated at allosteric sites and exosites. In order to target a specific caspase, group of caspases, or subset of caspase substrates, it is essential to understand the dif- ferences between individual caspases and the similarities within caspase subgroups. Thus, our long-term project goal has been to define and exploit unique regulatory features for each of the apoptotic caspases. By identifying allosteric sites and exosites, we have observed and described four major mechanistic classes of exosite and allosteric regulation. The first, shared by many disparate regulators, is the indirect disruption of the loops that cooperatively form the substrate-binging groove and impact catalysis. Based on our studies of caspase regulation via loop disruption, we developed an allosteric inhibitor that is more potent than any reported and is also by far the most selective, preferring caspase-6 by 500-fold over all other caspases. This selectivity is achievable because this new allosteric site is present exclusively in caspase-6. Given this success, we aim to investigate the remaining three classes of allosteric regulation. In Aim 1, we focus on class II, identifying exosites on caspase-6 and its substrates. This concerted analysis is possible for the first time due to our development of a hybrid caspase with the active site specificity of caspase-6 but the exosites of caspase-7. We aim to block particular exosites and explore the impact on a proteome-wide basis. We anticipate that this approach will enable the development of new inhibitors that block cleavage of disease-causing caspase-6 substrates like DJ-1,Tau or huntingtin in Alzheimer and Huntington but not other substrates. In Aim 2, we focus on class III, native small molecule binding. Our recent discovery that ATP binds to an orphan allosteric cavity and new methods will allow us to identify both covalent and non- covalent native ligands that regulate caspase-6 from this site. This goal is significant as it will provide need- ed insights into the intersection between caspases and metabolism. In Aim 3, we focus on class IV, which impact the folded state. We interrogate a caspase-9 site that when phosphorylated leads to disassembly of the core. This is the only site of phosphorylation that is conserved among all human caspases. Together this work has significant therapeutic implications in both proliferative and neurodegenerative diseases.